Gene therapy for the thalassemia syndromes and hemoglobinopathies requires that the introduced globin gene mimic the function of an in situ normal globin gene both in tissue specificity and efficiency of transcription. See e.g., Anderson, W. F. (1984) Science 226, 401-409. DNA mediated gene transfer experiments have shown that the human beta-globin gene with its immediate 5' and 3' flanking sequences can be expressed in a manner that is specific for the tissue and the stage of development. Chada, K. et al. (1985) Nature 314:377-380; Townes, T. et al. (1985) EMBO 4, 1715-1723; Kollias, G. et al. (1986) Cell 4b, 89-94; Rutherford, T. and Nienhuis, A. (1987) Mol. and Cell Biol. 7, 398-402; Constantini, F. et al. (1986) Science 233, 1192-1194; Magram, J. et al. (1985) Nature 315, 338-340. However, the transcriptional efficiency of such an introduced beta-globin gene is generally low (see references cited supra). A high level of transcription may require yet another level of cis control exerted by sequence elements, such as the enhancer sequences, that do not reside in the globin structural gene and its immediate flanking sequences (Kollias, G. et al., supra).
In the transgenic mouse, for example, low level but tissue-specific and even developmental-stage specific expression of the beta-globin gene has been flanking sequence was injected into the mouse zygotes. The expression level of the introduced beta-globin gene in erythroid cells of the transgenic mice is very low-less than a few percent of that of the endogenous mouse beta-globin gene. In a few transgenic mice, however, the m-RNA transcribed from the injected beta-globin gene has been reported to be as high as 50% of the endogenous mouse beta-globin mRNA (see Townes, T. et al. and Constantini, F. et al., supra.). In such cases, multiple copies (20-50 copies) of the injected beta-globin gene have been found to be integrated into the host chromosome. High level accumulation of the transcripts from these integrated genes may be the result of either the cumulative effect of many such integrated genes transcribed at low rates or of the chance integration of a copy of the introduced gene into an activated host chromosomal site. This position effect, i.e., the dependence of the expression level on the transcriptional activity of the site of integration, suggests that yet another level of cis transcriptional control located farther away from the gene and its immediate flanking sequences such as may possibly be exerted by an enhancer element, is required for efficient transcription of an introduced beta-globin gene.
Enhancer elements have been identified in the viral SV40 genome (Bernolst, C. and Chambon, P. (1981) Nature 290:309-310) and in the eukaryotic immunoglobulin gene cluster (See e.g., Gillies, S. et al. (1983) Cell 33, 717-728; Banerji et al. (1983) Cell 33, 729-740; Mercola, M. et al. (1983) Science 221, 663-665; Queen, C. and Baltimore D. (1983) Cell 33, 741-748; Picard, P. and Schaffner W. (1983) Nature 307 80-82; Gillies, S. and Tonegawa, S., U.S. Pat. No. 4,6763,281). They are capable of transcriptionally activating cis-linked genes over long distances, and their action is independent of the orientation of the enhancer element, and of its position with respect to the gene. In contrast to the SV40 viral enhancer which exhibits wide cell type specificity, the Ig enhancer and other identified eukaryotic enhancer elements display tissue specificity: the Ig enhancer appears to be most active in lymphoid cells (see references cited immediately above), the insulin enhancer element, in pancreatic beta cells (Edlum, T. et al. (1985) Science 230, 912), and an albumin enhancer, in liver cells (Pinkert, C. et al. (1987) Genes and Development 1, 268).
High level transcription of the transfected beta-globin gene can indeed be achieved by the presence in cis of the SV40 enhancer (see e.g., Banerji, J. et al. (1981) Cell 27, 299-308.) or the immunoglobulin (Ig) gene enhancer (Banerji, J. et al. (1983) Cell, 33:729-740). The tissue specific expression of such enhancer-beta globin gene constructs is, however, dictated by the host range of the cis enhancer, such that the beta-globin gene driven by the Ig enhancer is expressed most efficiently not in erythroid but in lymphoid cells (Banerji et al., (1983) Cell 33:729-740). This suggests not only that high level expression of the transfected beta-globin gene requires the presence of a cis enhancer but also that the tissue specific element contained in the enhancer can override the tissue specificity of the promoter.
The Friend Leukemia virus, which induces erythroleukemia, has been reported to contain erythroid specific enhancer elements in its long terminal repeats (LTR's) (Booze, Z. et al. (1986) EMBO 5, 1615-1623). Because of the carcinogenic property of the viral sequences, the applicability of this erythroid specific viral enhancer element to gene therapy appears limited. Although the existence of erythroid-specific enhancers has been postulated, no such enhancer has been identified in mammalian cells. Tuan et al. examined major DNase I-hypersensitive sites in the human "beta-globin-like" gene domain and showed that they were located in DNA regions which have certain characteristics of enhancers. (See Tuan, D. et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6384-6388; Tuan D. and London, I. M. (1984) Proc. Natl. Acad. Sci. USA 81, 2718-2722).
Retroviral vectors offer unique advantages over conventional methods for introduction of genes into host cells, such as hematopoietic cells, since they can be used to introduce an intact single copy of a gene into most mammalian cell types at much higher efficiencies, sometimes approaching 100%. (Weiss, R. et al. (1985) RNA Tumor Viruses, 2nd Ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.). Construction of enhancerless retroviral vectors in which the viral enhancer sequences are deleted from both the LTRS of the provirus, yields proviruses that are transcriptionally inactive, thus freeing the genomic insert in such vectors from the potential effects of vector transcription (Cone, R. et al (1987) Mol. and Cell Biol. 7, 87). The absence of enhancer sequences in both LTRs of the integrated provirus should also minimize the possibility of activating cellular proto-oncogenes and may thus provide a safer alternative in human gene therapy.
An enhancerless retroviral vector, which comprises: (1) an intact 5' LTR (2) a 3' LTR with a deletion in the retroviral enhancer sequence (3) a human beta-globin gene insert and (4) a selectable marker gene, the bacterial neomycin phosphotransferase gene (Neo.RTM.), has been constructed (Cone, R. et al., supra.). In the absence of a transcriptional enhancer element, the transduced human beta-globin gene, even though efficiently introduced by retroviral infection into the host cells, is inefficiently expressed, at a level about 500-fold less than the endogenous beta-globin gene (Cone et al., supra.) Another variant construct of the enhancerless retroviral vector contains the selectable Neo.RTM. gene and a c-myc oncogene coupled to the kappa immunoglobulin gene enhancer-promoter combination which confers B cell specific expression on cis linked genes (Dick, J. et al. (1985) Cell 42, 71). The c-myc gene in this vector was found to be transcribed in a B cell line infected with this recombinant retrovirus (Hawley, R. et al. (1987) Proc. Natl. Acad. Sci. 84, 2406).
Hematopoietic cells, infected with recombinant retroviruses, when injected into unirradiated or irradiated host animals, were found to be capable of long-term reconstitution of the marrow cells of the recipient animals. Hawley et al., supra, Lemishka, I. et al. (1986) Cell 45, 917.